Mitochondrial Creatine Kinase Is Not Required For Adp Transport Efficiency During Exercise

It is widely accepted that mitochondrial creatine kinase (Mi-CK) contributes to ~80% of ADP transport into mitochondria. However, this notion is based on calculated algorithms, and direct in vivo evidence to support the assumption that Mi-CK is a major regulator of mitochondrial ADP transport does not exist. PURPOSE: To elucidate the necessity of Mi-CK for in vivo ADP transport at rest and during exercise. METHODS: Mi-CK wild-type (WT) and knockout (KO) mice (n=6-8/group) were randomized to a sedentary (SED) or exercise (EX) group (acute bout; 1 hour, 15m/min, 5% grade). Resting RER and Western blotting of proteins involved in ADP/ATP transport (ANT1, ANT2), as well as cytosolic CK, Mi-CK, OXPHOS, COXIV, and PDHE1 were determined. All mice completed a run test to exhaustion. Muscle metabolites (ATP, phosphocreatine (PCr), creatine (Cr), glycogen, lactate) were determined in SED and EX mice. Permeabilized muscle fibres (PMFs) were used to examine Mi-CK contributions to ADP sensitivity in the presence and absence of Cr (20mM) in both SED and EX mice. RESULTS: The Mi-CK enzyme was not present in the mitochondria of KO mice, and while PMFs displayed an attenuated response to exogenous Cr, this response was not completely absent in KO mice. These data suggest solely analyzing ADP sensitivity in PMFs with Cr is confounded by the cytosolic CK enzyme, and therefore we relied on in vivo assessments of metabolism to determine the biological relevance of Mi-CK. There were no differences in SED VO2, RER, muscle metabolites, or any protein measured between WT and KO mice. Run time to exhaustion was similar between groups. Muscle PCr and glycogen decreased similarly in EX mice (WT and KO) relative to SED mice, along with an increase in Cr and lactate. In PMFs the apparent ADP sensitivity (Km) was not altered by exercise in WT mice, however, exercise decreased the apparent Km to ADP in KO mice. CONCLUSION: In contrast to previous estimates in the heart, the present data provides in vivo evidence that Mi-CK is not required for mitochondrial ADP respiratory sensitivity in resting muscle or during exercise. However, in situ experiments reveal that ADP sensitivity is increased in KO mice to optimize ADP transport and energy provision during exercise, suggesting either ANT or ATP synthase proteins are externally regulated.

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